Gallotannins and ellagitannins are members of the hydrolyzable tannin class of plant polyphenols. Tannins are secondary metabolites found throughout the plant kingdom that were first isolated and characterized in the 1950's. To date, over 500 ellagitannins and 200 gallotannins have been identified.
Gallotannins are the simplest hydrolyzable tannins. Compounds of this species consist of a carbohydrate core, usually glucose, which has been acylated with gallic acid. Variations among the gallotannins arise from differences in the stereochemistry at the anomeric carbon and the extent of galloylation about the carbohydrate core.
The more complex ellagitannins are comprised of the same building blocks as gallotannins. The defining characteristic of an ellagitannin is the presence of at least one hexahydrodiphenoyl moiety (HHDP), presumably formed by intramolecular oxidative C—C coupling between two galloyl groups. The HHDP units have been identified as bridging the 1,6-, 1,3-, 3,6- and 2,4-positions of the carbohydrate core, but are most often found between positions 2 and 3 or 4 and 6.
Ellagitannins can be either monomeric or oligomeric. Variations among simple ellagitannins include differences in the stereochemistry at the anomeric carbon and the number, positioning and stereochemistry of the HHDP units. Higher order tannins can be dimeric, trimeric, or tetrameric. The carbohydrate cores of oligomeric ellagitannins are joined by either a dehydrodigalloyl functionality most likely formed by intermolecular C—O oxidative coupling between two anomeric galloyl units, or by similar bonding between a galloyl group and an HHDP unit.
Lipopolysaccharide (LPS), or bacterial endotoxin, is a component of the cell wall in all gram-negative bacteria. The structure of LPS consists of four sections that are covalently linked: an O-specific chain which is comprised of oligosaccharides, an outer core and an inner core, made up of octulosonic acids and heptopyranoses, and a lipid membrane anchor, termed lipid A. Host immune cell response to LPS involves secretion of the cytokines interleukin-1β (IL-1β) and TNF-α. Overproduction of these cytokines, in particular TNF-α, can result in sepsis and septic shock.
Sepsis is caused by production of low levels (<1 ng/mL) of TNF-α as a result of exposure to LPS from a gram-negative bacterial infection. Characteristic symptoms of sepsis are hypothermia, fever and an increase in white blood cell count. Production of higher levels of TNF-α (>100 ng/mL) can result in the potentially lethal condition septic shock. Septic shock causes more than 20,000 deaths per year in the United States alone, and is the leading cause of death in intensive care units. This condition causes circulatory collapse, resulting in multiple organ failure and cardiovascular prolapse.
The lipid A portion of LPS mediates the endotoxic activity of the molecule. The process is initiated by bacterial lysis, resulting in liberation of LPS from the bacterial cell wall and exposure of lipid A. At low concentrations in the serum, LPS is bound by an LPS binding protein (LBP). This dimeric complex ligates to a membrane bound receptor on peripheral blood mononuclear cells' (PBMC's) CD14. CD14 must associate with (or otherwise activate) a second receptor identified as Tlr4 to initiate the signal transduction that results in cytokine release. At high concentrations of LPS, direct binding to a second membrane-bound receptor, L-selectin, is possible, which also results in cytokine release.
It is unknown how LPS interacts with LBP, or how the LPS/LBP complex ligates to CD14. It is know that synthetic and natural E. coli lipid A exhibit the highest endotoxicity compared to all other synthetic and natural lipid A samples. Attempts to identify the structural features of E. coli lipid A which impart the endotoxic activity have yielded negative results. Any variations in the structure of E. coli lipid A causes a decrease or lack of endotoxicity.
Currently, there are no widely effective treatments for septic shock. Current approaches to inhibiting LPS-induced bacterial sepsis include use of 1) LPS antagonists which presumably block the lipid A/receptor interactions; or 2) monoclonal antibodies which are designed to sequester various components of the septic shock response (LPS, TNF-α, TNF-α receptors, LPS receptors, etc.). The former strategy has generally relied on either lipid A analogs/derivatives which are quite potent, but so structurally complex as to render scale-up production problematic, or on small molecule agents which are more accessible but much less potent. The latter approach is hampered by cost concerns, and ultimately has been disappointing in in vivo efficacy trials.
Overproduction of TNF-α and other cytokines is also believed to underlie several debilitating diseases, such as leprosy, rheumatoid arthritis, and cachexia, the latter achieving notoriety in the context of late-stage AIDS. Consequently, inhibition of cytokine secretion has become the goal of numerous therapies.
Increasing TNF-α levels has been a focal point of numerous therapeutic regimes targeted at tumor remission. In the most favorable cases, administering relatively high concentrations of TNF-α directly to tumor sites has produced striking responses in patients with melanoma and sarcoma. Barbara et al. 1996. However, systemic application of TNF-α is an ineffective therapy as a consequence of its severe inflammatory effects (similar to IL-1β) and rapid clearance from serum (t1/2≈6.5-10.5 min). Sanches-Cantu et al. 1991.
The ellagitannin subfamily of the hydrolyzable tannins spans over 500 structurally characterized members. An increasing interest in the role played by these secondary plant metabolites in polyphenol-rich folk medicines from China and Japan has led to the identification of several ellagitannins which hold promise as potent antiviral and anticancer therapeutic agents. See e.g. Berlinck et al. (1995).
A number of oligomeric ellagitannins, including coriariin A and the structurally related species agrimoniin and gemin A have been found to induce tumor regression in mice infected with sarcoma-180 tumors through the increase of production of IL-1β. Miyamoto et al., Chem. Pharm. Bull. 1987 and Anticancer Res. 1993. The monomeric ellagitannins tellimagrandin I, tellimagrandin II, β-D-PGG and pedunculagin have been found to be much less effective antitumor agents.
The present inventors have now surprisingly discovered that certain gallotannins and ellagitannins play a role in either up-regulating and down-regulating the production of TNF-α and other cytokines. Some of these compounds have been found to be useful in decreasing secretion of TNF-α, thereby making them suitable in the development of treatments for diseases associated with overproduction of TNF-α, such as septic shock, leprosy, and cachexia. Other gallotannins have been found to be effective in increasing TNF-α levels for use in tumor remission.
Accordingly, it is a primary objective of the present invention to provide compositions and methods for regulating the production of cytokines, such as TNF-α and IL-1β using gallotannins and ellagitannins.
It is a further objective of the present invention to provide a composition and method for increasing levels of TNF-α and other cytokines to induce tumor remission using gallotannins and ellagitannins.
It is a further objective of the present invention to provide a composition and method for treating diseases associated with acute overproduction of cytokines, including IL-1β and TNF-α, such as in sepsis and septic shock, using gallotannins.
It is still a further objective of the present invention to provide a composition and method for treating diseases associated with chronic overproduction of lower levels of cytokines, including IL-1β and TNF-α, such as leprosy, rheumatoid arthritis, and cachexia, using gallotannins.
It is a further objective of the present invention to provide compositions and methods for regulating the production of TNF-α using gallotannins and ellagitannins that have low toxicity.
It is a further objective of the present invention to provide compositions and methods for regulating the production of TNF-α using gallotannins and ellagitannins that are effective in vivo.
It is yet a further objective of the present invention to provide compositions and methods for regulating the production of TNF-α and other cytokines using gallotannins and ellagitannins that are simple to synthesize and economical to manufacture.
It is yet a further objective of the present invention to provide compositions and methods for regulating the production of TNF-α and other cytokines using gallotannins which are LPS antagonists.
It is a further objective of the present invention to provide compositions and methods for regulating the production of TNF-α and other cytokines using gallotannins and ellagitannins which are LPS agonists.
It is still a further objective of the present invention to provide compositions and methods for regulating the production of TNF-α and other cytokines using gallotannins that induce little or no secretion of the cytokine IL-1β.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.